Chondrogenesis is the earliest well-orchestrated and controlled phase of skeletal development, involving mesenchymal cell recruitment and migration, condensation of progenitors, chondrocyte proliferation and differentiation, and maturation. This process is controlled exquisitely by cellular interactions with the growth factors, surrounding matrix proteins and other environmental factors that mediate cellular signaling pathways and transcription of specific genes in a temporal-spatial manner [1-3]. Production of and response to different growth factors are observed at all times and autocrine and paracrine cell stimulations are key elements of the process [4, 5]. Particularly relevant is the role of the TGF-beta superfamily, and more specifically of the BMP subfamily. Other factors include retinoids, FGFs, GH, and IGFs [6-8]. The growing evidences demonstrated that complicated cellular signaling language and informational content of chondrogenesis lie, not in an individual growth factor, but in the entire set of growth factors and others signals to which a cell is exposed [4, 5, 9]. The ways in which growth factors exert their combinatorial effects are becoming clearer as the molecular mechanisms of growth factors actions are being investigated. Gene- and cell-based therapy of growth factors for cartilage disorders are under intensive study. The isolation of the growth factor(s) that regulating chondrogenesis is therefore of great importance from both a pathophysiological and a therapeutic standpoint.
Granulin/epithelin precursor (GEP), a previously unrecognized growth factor in cartilage, was identified, for the first time, to be a novel chondrogenic factor based on the following findings: GEP is highly expressed in the chondrocytes in various differential stages of growth plate; GEP co-localizes with COMP, a major component of cartilage, in the primary adult chondrocyte and these two proteins bind to each other; GEP, whose level is strongly upregulated by both chondrogenic growth factors (BMP-2 and TGF-beta) and proinflammatory cytokines TNF-alpha, promoted proliferation and chondrocyte differentiation from stem cells; and GEP affects chondrocyte functions.
In the progression of arthritis, synovium, cartilage and bone are each sites of increased growth factor, cytokine, and inflammatory mediator production that are believed to contribute to disease pathogenesis [10, 11]. Although both bone and synovium have important roles in the pathogenesis of arthritis [10, 12], most effort in disease modifying treatments has focused on molecular events within articular cartilage. Arthritic chondrocytes undergo a series of complex changes, including hypertrophy, proliferation, catabolic alteration and, ultimately, death. The regulation of these phenotypic changes at different stages of disease is also under intensive study, with focus on the biomechanical and biochemical signals that regulate each of these discrete chondrocyte responses [11, 13]. Chondrocytes themselves are featured protagonists in this cascade of change, not only the target of external biomechanical and biochemical stimuli, but also themselves the cellular source of cytokines, chemokines, proteases and inflammatory mediators that promote the deterioration of articular cartilage [10, 11]. Pathogenic molecules produced by arthritic chondrocytes include matrix metalloproteinases (MMPs), ADAMTSs, interleukin (IL)-1, tumor necrosis factor (TNF), IL-6, IL-8, nitric oxide, prostaglandins and leukotrienes [11, 13]. There is also evidence that arthritic chondrocytes exhibit increased anabolic activity, including increased release of growth factors and synthesis of type II collagen, proteoglycan, extracellular matrix protein 1 (ECM1, our unpublished data) and other extracellular matrix proteins, as well as the expression of genes associated with the chondroprogenitor hypertrophic phenotype [14-16].
GEP, also known as PC-cell-derived growth factor (PCDGF), progranulin (pgrn), proepithelin, acrogranin, GP80, was first purified as a growth factor from conditioned tissue culture media [17, 18]. It has been identified from different sources by several independent laboratories [19-22]. GEP is a 593-amino-acid secreted glycoprotein with an apparent molecular weight of 80 kDa [19, 23], which acts as an autocrine growth factor. GEP contains seven and a half repeats of a cysteine-rich motif (CX5-6CX5CCX8CCX6CCXDX2HCCPX4CX5-6C) in the order P-G-F-B-A-C-D-E, where A-G are full repeats and P is the half motif (FIG. 1). The C-terminal region of the consensus sequence contains the conserved sequence CCXDX2HCCP and is suggested to have a metal binding site and to be involved in regulatory function [24]. Notably, GEP undergoes proteolytic processing with the liberation of small, ˜6 kDa repeat units known as granulins (or epithelins), which retain biological activity [25]: peptides are active in cell growth assays [22] and may be proinflammatory [26].
GEP is abundantly expressed in rapidly cycling epithelial cells, in cells of the immune system and in neurons [19-21, 26]. High levels of GEP expression are also found in several human cancers, and contributes to tumorigenesis in diverse cancers, including breast cancer, clear cell renal carcinoma, invasive ovarian carcinoma, glioblastoma, adipocytic teratoma, multiple myeloma [25, 27-33] and osteosarcoma (our unpublished data). Although GEP mainly functions as a secreted growth factor, it was also found to be localized inside the cells and directly modulate intracellular activities [21, 34-36]. The role of GEP in the regulation of cellular proliferation has been well characterized using mouse embryo fibroblasts derived from mice with a targeted deletion of the insulin-like growth factor receptor (IGF-IR) gene (R− cells). These cells are unable to proliferate in response to IGF-I and other growth factors (EGF and PDGF) necessary to fully progress through the cell cycle [37]. In contrast, GEP is the only known growth factor able to bypass the requirement for the IGF-IR, thus promoting growth of R− cells [22, 38]. Increasing evidences have also implicated GEP in the regulation of differentiation, development and pathological processes. It has been isolated as a differentially-expressed gene from mesothelial differentiation [39], sexual differentiation of the brain [40], macrophage development [41] and synovium of rheumatoid arthritis and osteoarthritis [42]. Remarkably, GEP was also shown to be a crucial mediator of wound response and tissue repair [30, 43]. Very recently, it was reported that mutations in GEP cause tau-negative frontotemporal dementia linked to chromosome 17 [44-47].
The mode of action of GEP remain largely unknown. Granulin binding sites have been demonstrated, although cell surface receptors have not yet been characterized [28, 48]. GEP leads to activation of the mitogen-activated protein kinase pathway and to stimulation of cyclin D1 protein expression. This can account for the cellular proliferation activity of granulin and its ability to replace estrogen in inducing the growth of breast cancer cells [49]. Several GEP-associated partners have been reported and found to affect GEP action in various processes. One example of this is the secretory leukocyte protease inhibitor (SLPI). Elastase digests GEP exclusively in the interepithelin linkers resulting in the generation of granulin peptides, suggesting that this protease may be an important component of a GEP convertase. SLPI blocks this proteolysis either by directly binding to elastase or by sequestering epithelin peptides from the enzyme [43]. It was found that GEP can modulate transcription activities by interacting with human cyclin T1, a component of positive transcription elongation factor b (P-TEFb) [35] and Tat-P-TEFb [34]. GEP was also found to interact with perlecan, a heparan sulfate proteoglycan and perlecan-null mice exhibit the severe skeletal defects [50-52]. The perlecan-GEP interaction was suggested to modulate tumor growth [28]. Our global screen led to the isolation of GEP as a novel binding growth factor of COMP, a noncollagenous component of the cartilage matrix. The interaction between these two molecules appears to regulate chondrocyte proliferation.
Modern methods of global analysis of protein-protein interactions followed by biological assessment have led to new ways to identify novel proteins not previously associated with the pathogenesis of a particular disease or organ system. Initially identified through a functional genetic screen, this application details the discovery that GEP, previously unknown as a growth factor in cartilage, is a novel mediator in chondrogenesis and arthritis. This extends our understanding of the actions of growth factors in cartilage biology and their application to treatment of cartilage disorders and arthritic conditions. The identification and manipulation of growth factors that regulate the chondrogenic potential of mesenchymal stem cells (MSCs), chondrocyte progenitors and chondrocytes can be used to optimize the therapeutic application of growth factors and these cells in cartilage disorders and connective tissue disorders.    1. 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The citation of references herein shall not be construed as an admission that such is prior art to the present invention.